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Structural Insights into Early Spliceosome Assembly by cryo-EM

dc.contributor.advisorStark, Holger Prof. Dr.
dc.contributor.authorZhang, Zhenwei
dc.date.accessioned2021-12-23T11:44:23Z
dc.date.available2022-01-06T00:50:08Z
dc.date.issued2021-12-23
dc.identifier.urihttp://hdl.handle.net/21.11130/00-1735-0000-0008-59D4-5
dc.identifier.urihttp://dx.doi.org/10.53846/goediss-9005
dc.identifier.urihttp://dx.doi.org/10.53846/goediss-9005
dc.language.isoengde
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subject.ddc572de
dc.titleStructural Insights into Early Spliceosome Assembly by cryo-EMde
dc.typecumulativeThesisde
dc.contributor.refereeStark, Holger Prof. Dr.
dc.date.examination2021-12-08
dc.description.abstractengIn eukaryotes, splicing is a process during which the non-coding introns of the pre-messenger RNA (pre-mRNA) are removed, and the protein-coding exons are ligated. The introns are defined by conserved 5' splice site (5'SS), 3'SS, and the branch site. These sequences are recognized by a highly dynamic molecular machinery called the spliceosome that catalyzes the splicing reactions. The spliceosome is a large ribonucleoprotein (RNP) complex that contains five small nuclear ribonucleoproteins (snRNPs), namely U1, U2, U4, U5, and U6 snRNPs, and other non-snRNP proteins. The spliceosome is assembled in a stepwise manner on the pre-mRNA, and during the early assembly phase, U1 and U2 snRNPs recognize the 5'SS and the branch site, respectively, forming the prespliceosome complex (A complex). In the A complex, the U2 snRNA base-pairs with the branch site, forming the U2-BS helix. The branch-site adenosine (BS-A), the nucleophile of the first catalytic step of splicing, is bulged out from the U2-BS helix and thereby defined. The U2-BS helix is stabilized by U2 proteins, especially the SF3b complex. As revealed by recent cryo-EM structures of fully assembled spliceosome complexes, SF3B1 (human)/Hsh155 (yeast), the major scaffolding protein of the SF3b complex, contains a ring-like HEAT domain and tightly wraps around the U2-BS helix. The closed conformation of the SF3B1/Hsh155 HEAT domain (SF3B1/Hsh155HEAT) found in the spliceosomes is distinct from the open conformation found in the crystal structure of isolated SF3b complex, but the trigger to this functionally important conformational change is unknown. The U2 snRNP also contains TAT-SF1 (Cus2 in yeast), whose function is only poorly understood. In yeast, U2 snRNA nucleotides that base pair with the branch site are initially sequestered in a branchpoint-interacting stem-loop (BSL), but it is unknown whether the human U2 snRNA folds in a similar manner. The A complex formation requires the DEAD-box ATPase Prp5 that has been shown to mediate an ATP-dependent conformational change in U2 snRNP. However, the Prp5 mediated RNP remodelling is poorly understood due to the lack of structural information of early spliceosomal complexes, especially in humans. Furthermore, Prp5 is also implicated in proofreading the branch site during the A complex formation. Charles Query's and Soo-Chen Cheng's lab have shown that branch-site mutations -- including a U to A mutation at position 257 (U257A) of the actin pre-mRNA, which destabilize the U2-BS helix -- hinder splicing reaction and lead to an accumulation of spliceosomes at an early assembly stage, at which Prp5 is still associated. Several mutations in Prp5 have been shown to improve the splicing efficiency of the pre-mRNAs containing branch-site mutations. This has led to the proposal that Prp5 functions in assessing the fidelity of U2 base-pairing with the branch site. However, the exact mechanism of how Prp5 proofreads the branch site is unknown. In the first part of this thesis, we report the first structure of the human 17S U2 snRNP by single-particle cryo-EM at a core resolution of 4.1 A and its molecular architecture based on crosslinking mass spectrometry (CXMS) data. Our 17S U2 snRNP structure shows that SF3B1HEAT adopts the open conformation, similar to the conformation identified in the crystal structure of isolated SF3b core and that the SF3b core complex does not undergo major structural changes during U2 assembly. The open conformation of SF3B1HEAT is likely stabilized by the binding of U2 proteins Prp5 and TAT-SF1. Our studies further reveal that U2 snRNA forms a BSL in humans, which is sandwiched by U2 proteins, including Prp5, TAT-SF1, and SF3B1, and is inaccessible for branch site recognition. Therefore, substantial remodelling of the BSL nucleotides and displacement of BSL interacting proteins must occur for stable U2 addition into the spliceosome. By comparing the 17S U2 structure with the U2 region of later spliceosome complexes, we reveal the structural rearrangements facilitated by Prp5 that are required for stable U2-BS interaction and the A complex formation. In the second part of this thesis, we investigate the molecular mechanism of how Prp5 contributes the branch site proofreading in yeast. We report cryo-EM structures of the yeast spliceosome intermediates assembled on actin pre-mRNAs with the BS-A deleted (Delta BS-A complex) or with the U257A mutation (U257A complex) at the branch site. We show that the two complexes are structurally identical at the current resolution, and they represent a novel spliceosomal intermediate, the pre-A complex, which is formed after Prp5 mediated U2 remodelling but prior to A complex formation. Our pre-A complexes reveal that formation of the U2-BS helix alone is not sufficient to trigger the closure of Hsh155HEAT. Instead, insertion of the BS-A into the SF3b binding pocket is the major trigger. A comparison of our pre-A complex structures with the 17S U2 snRNP and the A complex structures reveals that the U2 snRNP undergoes a large-scale remodelling during the U2 to pre-A complex and pre-A to A complex transition. Importantly, the pre-A to A transition, which generates the U4/U6.U5 tri-snRNP binding site, is inhibited by Prp5 binding. This provides a structural explanation of why Prp5 and the tri-snRNP binding are mutually exclusive, as previously observed. Our data also suggest that the displacement of Prp5 is coordinated with the docking of the U2-BS helix and the Hsh155HEAT closure that is trigger by the BS-A insertion. Branch-site mutations that hinder the correct insertion of the BS-A inhibit formation of the productive closed conformation of Hsh155HEAT and the release of Prp5, thus being stalled at the pre-A stage. Taken together, we propose that Prp5 does not proofread the branch-site sequence directly but rather the overall RNP conformation of the pre-A complex.de
dc.contributor.coRefereeUrlaub, Henning Prof. Dr.
dc.contributor.thirdRefereeLührmann, Reinhard Prof. Dr.
dc.contributor.thirdRefereeTittmann, Kai Prof. Dr.
dc.contributor.thirdRefereeFaesen, Alexis Caspar Dr.
dc.contributor.thirdRefereeStein, Alexander Dr.
dc.subject.engsplicingde
dc.subject.engspliceosomede
dc.subject.engU2 snRNPde
dc.subject.engPrp5de
dc.subject.engearly spliceosome assemblyde
dc.subject.engTAT-SF1de
dc.subject.engU1 snRNPde
dc.subject.engDEAD-box RNA helicasede
dc.subject.engproofreadingde
dc.subject.engSF3bde
dc.subject.engSF3B1de
dc.subject.engHsh155de
dc.identifier.urnurn:nbn:de:gbv:7-21.11130/00-1735-0000-0008-59D4-5-5
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de
dc.description.embargoed2022-01-06
dc.identifier.ppn1783650826


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